Deficiency in Opu Systems Imparts Salt-Sensitivity to Weizmannia coagulans

Weizmannia coagulans can be used as a starter strain in fermented foods or as a probiotic. However, it is salt-sensitive. Here, W. coagulans genomes were compared with the genomes of strains of Bacillus species (B. licheniformis, B. siamensis, B. subtilis, and B. velezensis) that were isolated from fermented foods and show salt tolerance, to identify the basis for the salt-sensitivity of W. coagulans. Osmoprotectant uptake (Opu) systems transport compatible solutes into cells to help them tolerate osmotic stress. B. siamensis, B. subtilis, and B. velezensis each possess five Opu systems (OpuA, OpuB, OpuC, OpuD, and OpuE); B. licheniformis has all except OpuB. However, W. coagulans only has the OpuC system. Based on these findings, the opuA and opuB operons, and the opuD and opuE genes, were amplified from B. velezensis. Expression of each of these systems, respectively, in W. coagulans increased salt-tolerance. W. coagulans expressing B. velezensis opuA, opuD, or opuE grew in 10.5% NaCl (w/v), whereas wild-type W. coagulans could not grow in 3.5% NaCl. The salt resistance of B. subtilis was also increased by overexpression of B. velezensis opuA, opuB, opuD, or opuE. These results indicate that the salt-susceptibility of W. coagulans arises because it is deficient in Opu systems.


Introduction
Bacillus coagulans was first isolated from spoiled evaporated milk [1] and later reclassified as Weizmannia coagulans based on genomic analysis [2].W. coagulans is a Gram-positive, lactic acid-producing, facultative anaerobic, spore-forming bacterium.It is able to endure extreme conditions such as high-temperature, food processing, stomach acids, bile, and high salt, in the form of spores.These properties are great advantages for a probiotic strain that must go through the gastrointestinal tract and an industrial strain that must go through food processing steps [3].
W. coagulans has been studied for a long time as a probiotic bacterium for humans [4][5][6].It has several health benefits, including prevention of muscle damage during exercise, improvement of gastrointestinal disorders, ease of diarrhea, and prevention of bacterial vaginosis [4][5][6].Health benefits of W. coagulans have been demonstrated in human studies [7,8], and W. coagulans is sold commercially as a probiotic.
W. coagulans is naturally distributed in a variety of niches including soil, water, and air.It is also found in food materials such as soybean, locust bean, maize, rice, and others [3,[9][10][11].W. coagulans produces exo-enzymes such as amylase, protease, and lipase.These activities contribute to improvement of the quality and sensory properties of fermented foods via protein and carbohydrate degradation [3,12,13].W. coagulans also produces compounds with activity against Gram-positive and Gram-negative bacteria [14].Therefore, W. coagulans is used in various commercial food products, including dairy foods, fermented meats, cereals, baby foods, and ice cream [3].
The Qualified Presumption of Safety (QPS) system of the European Union Food Safety Authority was introduced to assess the safety of food and feed microorganisms [15], and W. coagulans has been on the QPS list since 2007.Also, four W. coagulans strains have been given Generally Recognized as Safe status by the US Food and Drug Administration (http://www.fda.gov/)[16].W. coagulans is also on the Food Materials list of the Ministry of Food and Drug Safety, Korea (Notification of the Ministry of Food and Drug Safety No. 2024-20).These classifications confirm that W. coagulans is nonpathogenic and can be applied in the food industry, including as a probiotic.
Most research on W. coagulans has focused on its functional properties as a probiotic strain, while its use as a starter candidate for fermented foods has been studied less.However, 31 W. coagulans strains from rice straw were assessed for their antibiotic susceptibility using the minimum inhibitory concentration test and their enzymatic (protease and lipase) activities, to select safe starter candidates for sensory improvement of fermented foods [17].At the same time, it was found that W. coagulans did not grow on tryptic soy agar (TSA) containing 3% NaCl (w/v).The NaCl concentration of most fermented foods is >2%, and for starters to work efficiently in fermented foods, Weizmannia coagulans can be used as a starter strain in fermented foods or as a probiotic.However, it is salt-sensitive.growth must be possible in the salt concentration of the food.Here, we determined the reason for the saltsensitivity of W. coagulans.

Bacterial Strains and Culture Conditions
The strains and plasmids used in this study are listed in Table 1.Escherichia coli was cultured in Luria-Bertani agar (Becton, Dickinson and Co., USA), Bacillus in tryptic soy agar (TSA; Becton, Dickinson and Co.), and W. coagulans in de Man, Rogosa, and Sharpe (Becton, Dickinson and Co.) agar.When necessary, antibiotics were added to the growth medium at the following concentrations: ampicillin, 100 μg/ml; erythromycin, 10 μg/ml; kanamycin, 10 μg/ml.

Comparative Genomic Analyses
For comparative genomic analysis of W. coagulans and four Bacillus species, which were mainly isolated from fermented foods, genome sequence data were obtained from the NCBI (http://ncbi.nlm.nih.gov/genomes)(Table 2).Genes were predicted using the RAST server for SEED-based automated annotation [40].The predicted genes of the strains were confirmed using CLgenomics ver.1.55 software (CJ Bioscience, Republic of Korea) and the iPath (ver.3) module [41].

DNA Cloning and Transformation
Plasmids and genomic DNA of E. coli, B. subtilis, and W. coagulans were extracted with an Inclone Plasmid Mini Prep Kit (Inclone Biotech, Republic of Korea) and a DNeasy Tissue Kit (Qiagen, Germany), respectively, according to the manufacturers' instructions.B. subtilis and W. coagulans were treated with lysozyme (0.1 g/ml) at 37°C for 1 h before DNA extraction.
Genes related to the Opu system from B. velezensis DMB07 were amplified by PCR using primer sets containing   the appropriate restriction enzyme sites for insertion into pLipSM or pYJ335 (Table 3).PCR amplifications were performed using a T3000 thermocycler (Biometra, Germany) [42] and an Inclone Taq Polymerase Kit (Inclone Biotech) according to the manual.PCR primers were designed based on Opu system genes and nearby sequences of B. velezensis strain DMB07 (Table 3).All PCRs were performed using 30 cycles of denaturing at 95°C for 1 min, annealing at 63°C for 1.5 min, and elongation at 72°C for 1.5 min.Amplicons were digested by appropriate enzymes and inserted into same sites of pLipSM or pYJ335.Constructed plasmids were verified by PCR using pLipSM-check-F primer or pYJ335-check-F primer and DNA sequencing.Constructed plasmids derived from pYJ335 were introduced into E. coli DH5α (Stratagene, USA) by the method of Hanahan and Meselson [43], and then into W. coagulans KCTC 3625 T (KCTC; Korean Collection for Type Cultures, South Korea) by electroporation [44] with a gene pulser (Bio-Rad, USA).For introduction into B. subtilis ISW1214 of pLipSM-derived plasmids, DNA extracted from E. coli BL21 (DE3) (New England Biolabs, USA) was used [20].

Determination of Salt Tolerance
Salt tolerance of the strains in this study was determined by examining growth on TSA supplemented with up to 14% NaCl (w/v, final concentration).Growth on 0.5% (the NaCl concentration in normal TSA), 3.5%, 7%, 10.5%, and 14% NaCl was determined after incubation for 1, 2, 3, 4, and 5 days.The experiment was performed three times, independently.

Growth of W. coagulans on TSA
W. coagulans originally belonged to the genus Bacillus but was later reclassified.Most Bacillus species can grow on medium supplemented with >7% NaCl (w/v, final concentration) [45][46][47].In contrast, W. coagulans does not grow at a salt concentration of >3% [17].To reconfirm this, the growth of W. coagulans was checked in different salt conditions.As shown in Fig. 1, W. coagulans grew well on TSA without added extra salt (TSA contains 0.5% NaCl), while it did not grow on TSA with final NaCl concentrations of 3.5% or 7%.In contrast, B. licheniformis, B. siamensis, B. subtilis, and B. velezensis grew on TSA with a final NaCl concentration of 7% (Fig. 1).

Comparative Genomic Analysis of Salt Tolerance
B. licheniformis, B. siamensis, B. subtilis, and B. velezensis are predominantly isolated from fermented soybean products such as meju and doenjang [45,48].The enzymatic activities of these species contribute to the development of sensory properties of the fermented foods.Therefore, several studies have reported analysis of these strains for use as starters [38,49,50].W. coagulans is also isolated from several foods and exhibits protease and lipase activities, so it was judged that it could improve the sensory properties of fermented foods.However, W. coagulans is sensitive to salt compared with the four Bacillus species.Therefore, we undertook comparative genomic analysis of these species, looking for factors that affect their salt tolerance (Table 2).
To resist osmotic stress, many bacteria accumulate compatible solutes such as choline, glycine betaine, and proline betaine through uptake from outside the cell via transporters including osmoprotectant uptake (Opu) transporter [51,52].The Opu system is known to be involved in salt resistance [51].W. coagulans genomes possess the OpuC system.Meanwhile, B. licheniformis encodes four Opu systems, and B. siamensis, B. subtilis, and B. velezensis possess five Opu systems (Fig. 2).OpuA, OpuB, and OpuC belong to the ATP-binding cassette transporter superfamily, and OpuD and OpuE are single-component transporters belonging to the BCCT (Betaine/Carnitine/Choline Transporters) family [51].In the current study, the encoded OpuA, OpuB, and OpuC systems were found to contain the common substrate-binding, transporter, and ATP-binding proteins.Although Hoffmann and Berner reported that Bacillus species contain five Opu systems [51], B. licheniformis genomes lack the OpuB system.Our results suggest that the low salt-tolerance of W. coagulans compared with high salttolerance of Bacillus species is due to it only having one Opu system.

Opu Systems Enhance the Salt Tolerance of B. subtilis
As a result of the comparative genome analysis, the salt-sensitivity of W. coagulans was reasoned to arise from a deficiency in Opu systems.To test that, we aimed to introduce "absent" genes into W. coagulans to determine the effect on salt resistance.Plasmid pLipSM is an E. coli/Bacillus shuttle vector [20].Although W. coagulans has been reclassified from genus Bacillus, this plasmid is also functional in Weizmannia.opu genes from B. velezensis DMB07 (which contains all the opu genes and had the highest salt resistance among the strains we tested) were cloned and introduced into W. coagulans via this plasmid.Note that we did not clone opuC from B. velezensis DMB07 because W. coagulans naturally possesses this system (see section 2.2).The obtained recombinant plasmids were designated pL-opuA, pL-opuB, pL-opuD, and pL-opuE, respectively.In addition, pL-opurB was reconstructed with the opuB operon and the yvaV gene, which is a regulator for expression of the opuB operon (Fig. 3A).
Because W. coagulans strain KCTC 3625 T is a wild-type, it was not expected to be easy to introduce recombinant plasmids into the strain, so they were first introduced into laboratory strain B. subtilis ISW1214.We tested whether the protein expression vectors had an effect on the salt resistance of transformed B. subtilis ISW1214.As shown in Fig. 3B, wild-type B. subtilis ISW1214 grew in medium containing 7% NaCl, but not in medium containing 10.5% or 14% NaCl.However, when any one of the B. velezensis Opu systems was overexpressed, the resulting strain grew on 14% NaCl.The multicomponent systems OpuA and OpuB enabled better growth on 14% NaCl than the single-component systems OpuD and OpuE (Fig. 3B).

Opu Systems Confer Salt Tolerance to W. coagulans
pLipSM-based plasmids produced in B. subtilis were purified and introduced into W. coagulans KCTC 3625 T , but W. coagulans KCTC 3625 T showed resistance to low concentrations of kanamycin, which is the selection marker in pLipSM, so it was not easy to select W. coagulans transformants containing pLipSM-derived plasmids.Accordingly, we tried to introduce opu genes into W. coagulans KCTC 3625 T by using pYJ335, which has the erythromycin resistance gene as a selection marker.The replicon of plasmid pYJ335 was derived from pUC19 and pE194 fragments [21].The replicon can be cloned from lactic acid bacteria and Staphylococcus [53].The opu genes were amplified from B. velezensis DMB07 in the same way as in the previous experiment (see section 3.3), and recombinant plasmids were generated by inserting the opuA and opuB operons, and opuD and opuE genes, to produce pYJ-opuA, pYJ-opuB, pYJ-opuD, and pYJ-opuE (Fig. 4A).The vector containing the opuB operon, including the yvaV gene, which is a regulator for expression of the opuB operon, was named pYJ-opurB.These vectors were each transformed directly into W. coagulans (without going through B. subtilis).All the transformed strains grew in medium with 7% NaCl added, and all except the OpuB-containing transformants grew in 10.5% salt (Fig. 4B).
In previous studies, 67.7% and 71.0% of W. coagulans strains showed protease and lipase activities, respectively; these activities produce amino acids and fragrance components from lipids, respectively [17].In addition, it can be predicted that all the strains of W. coagulans have production performance in the condition containing NaCl, and that most strains can inhibit the growth of other microorganisms via antibacterial activity [17] However, the W. coagulans strains were sensitive to salt.Most fermented foods contain a significant salt content.Therefore, even though W. coagulans is widely distributed in the environment [3, 9-11, 54, 55] and in the raw materials used to prepare fermented foods, detection of this species is lowered in foods with added salt [17].If W. coagulans were to be salt-resistant, it would be expected to play a greater role in fermented food production.Here, the genetic basis of the sensitivity of W. coagulans to salt, i.e. a deficiency in Opu systems, was determined.These results will help with screening for salt-resistant strains.

Fig. 2 .
Fig. 2. Predicted mechanism of salt-sensitivity of W. coagulans based comparative genomic analysis with four Bacillus species.Compatible solutes are depicted in black text in yellow boxes.Color coding indicates which species contain which opu genes.

Fig. 3 .
Fig. 3. Construction of pLip-SM-derived plasmids containing opu genes from B. velezensis DMB07 (A) and salt-tolerance of B. subtilis containing these plasmids (B).TSA containing up to 14% NaCl (w/v, final concentration) was used for the detection of growth.

Fig. 4 .
Fig. 4. Construction of pYJ335-derived plasmids containing opu genes from B. velezensis DMB07 (A) and salt-tolerance of W. coagulans containing these plasmids (B).de Man, Rogosa, and Sharpe medium containing up to 10.5% NaCl (w/v, final concentration) was used for the detection of growth.

Table 2 . Genomic features of strains of Weizmannia coagulans and four Bacillus species.
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Table 3 . Oligonucleotides used in this study.
aRestriction sites are underline.